Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-10-02
2001-10-02
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S501000, C536S025300
Reexamination Certificate
active
06297008
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a chemiluminescence-utilizing process for detecting or quantifying a target nucleic acid, and a pyrylium compound used for chemiluminescence analysis.
2. Description of the Related Art
Nowadays, detection of double-stranded nucleic acids or specific genes, i.e. single-stranded nucleic acids having specific base sequences, is routinely carried out in various fields such as medicine, criminal investigations, and agriculture (hereinafter, such double- or single-stranded nucleic acids are referred to as target nucleic acids).
Double-stranded nucleic acids in samples have been detected, for example, as follows: A double-stranded nucleic acid is separated by electrophoresis using a polyacrylamide gel or an agarose gel; the resultant is then stained with a fluorochrome which can be intercalated between adjacent base pairs in the double-stranded nucleic acid to exhibit enhancement of fluorescence, such as ethidium.bromide (EB); the fluorochrome intercalated into the double-stranded nucleic acid is excited by a transilluminator with an ultraviolet lamp; and the fluorescence emitted from the fluorochrome is detected.
Similarly, a double-stranded nucleic acid in a solution can be detected by staining the double-stranded nucleic acid with a fluorochrome such as EB, diamidinodiphenylindole dihydrochloride (DAPI) or Hoechst 33258, and detecting fluorescence emitted from the fluorochrome.
A problem with the detection of a double-stranded nucleic acid in a solution using an ordinary fluorochrome whose fluorescence is enhanced by being associated with the double-stranded nucleic acid is low detection sensitivity in many cases. Fluorescence detection itself is more sensitive than more conventional colorimetry. The absolute sensitivity-limit of fluorescence detection, however, falls within a magnitude in the order of a few nM due to problems inherent in fluorescence measurement, such as leaking light derived from excitation light, and Raman scattering light from the solvent molecules when the sample is liquid. In particular, the sensitivity of fluorescence detection is not satisfactory for directly detecting a trace-amount or low-concentration of double-stranded nucleic acid derived from an organism.
Further, another problem with the detection of a double-stranded nucleic acid using a fluorochrome is the rise of the background during the detecting step due to fluorescence emission from the free fluorochrome molecules not associated with the double-nucleic acid when irradiated with ultraviolet rays. Such raised background can be a cause of lowered detection sensitivity.
As a remedy to solve the problem concerning the rise of the background, Japanese Patent Laid-Open No. 7-174759 (corresponding to U.S. Pat. No. 5,624,798) discloses a method using a pyrylium compound which has a specific structure and exhibits fluorescence only when it is associated with a double-stranded nucleic acid. According to this method, the detection sensitivity has been markedly improved, and detection utilizing fluorescence has markedly become practicable.
Although the detection sensitivity has been improved by reducing the level of the background, the above-described problems inherent in detection utilizing fluorescence, such as leakage of light and generation of Raman scattering light, has not yet been sufficiently solved. Accordingly, there has been a demand for a method to further improve the detection sensitivity.
Meanwhile, single-stranded nucleic acids having specific base sequences have been detected, for example, by a so-called probe method using a labelled nucleic acid as a probe. Various probe methods have been developed, in which radioisotopes, bioluminescent techniques or chemiluminescent techniques are employed to achieve high sensitivity.
In probe methods using radioisotopes, labels containing radio active atoms (radioisotopes) are used, and the detection sensitivity is satisfactory, and theoretically even one molecule (one copy) of the target nucleic acid can be detected. Such probe methods using radioisotopes, however, require special facilities, and the operation is accompanied by dangers. Further, since radioisotopes are unstable, probe nucleic acids labelled with radioisotopes cannot be stably stored for long time periods.
In contrast, probe methods using conventional chemical staining methods or enzymatic staining methods are more practical since they do not require special facilities, and the operation is relatively safe. Such probe methods using conventional staining methods are, however, markedly inferior in sensitivity to those using radioisotopes, and cannot sufficiently cope with detection of nucleic acids which can be obtained only in extremely small quantities, such as nucleic acids derived from organismal samples. Further, in many cases, probe nucleic acids bonded with labelling substances for such conventional staining methods are also unstable, and cannot be stored for long time periods.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a sensitized process for detecting a target single-stranded nucleic acid in a sample.
Another object of the present invention is to provide a process for more precisely quantifying a target single-stranded nucleic acid in a sample.
Further, another object of the present invention is to provide a process for more sensitively detecting a target double-stranded nucleic acid in a sample.
Further object of the present invention is to provide a more precise process for quantifying a target double-stranded nucleic acid in a sample.
Moreover, another object of the present invention is to provide a highly sensitive process for detecting a single-stranded nucleic acid in a sample without complicated procedures.
Furthermore, another object of the present invention is to provide a process for highly precisely quantifying a target single-stranded nucleic acid in a sample without complicated procedures.
Furthermore, another object of the present invention is to provide a highly sensitive process for detecting a double-stranded nucleic acid in a sample with a simple procedure.
Moreover, another object of the present invention is to provide a process for highly precisely quantifying a target double-stranded nucleic acid in a sample.
Still further, another object of the present invention is to provide a compound for use in a chemiluminescence analysis.
According to an aspect of the present invention, there is provided a process for detecting a target single-stranded nucleic acid having a first base sequence comprising the steps of:
forming a double-stranded nucleic acid by hybridizing the target single-stranded nucleic acid with a probe nucleic acid having a second base sequence complementary to the first base sequence;
providing a chemiluminescent compound capable of being associated with a double-stranded nucleic acid, and then associating the chemiluminescent compound with the double-stranded nucleic acid resulting from the above forming step; and
detecting luminescence from the chemiluminescent compound associated with the double-stranded nucleic acid.
According to another aspect of the present invention, there is provided a process for quantifying a target single-stranded nucleic acid having a first base sequence comprising the steps of:
forming a double-stranded nucleic acid by hybridizing the target single-stranded nucleic acid with a probe nucleic acid having a second base sequence complementary to the first base sequence;
providing a chemiluminescent compound capable of being associated with a double-stranded nucleic acid, and then associating the chemiluminescent compound with the double-stranded nucleic acid resulting from the above forming step; and
measuring luminescence from the chemiluminescent compound associated with the double-stranded nucleic acid.
According to these aspects, since the sensitivity of the detection of the target nucleic acid is quite high, it is not necessary to employ a target nucleic acid-amplifying process, such as PCR process.
Okamoto Tadashi
Yamamoto Nobuko
Canon Kabushiki Kaisha
Fitzpatrick ,Cella, Harper & Scinto
Marschel Ardin H.
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